Field of the Invention
The present invention relates to the technical field of microfluids, in particular to microfluids in combination with semiconductor technology. In particular, the present invention relates to a method for producing a plurality of measurement regions on a chip.
The present invention moreover relates to a chip having a multiplicity of electrically addressable measurement regions.
Description of Related Art
Microfluid systems are outstandingly suitable for carrying out chemical and biological detection reactions with low sample volumes.
In the field of clinical chemistry, in particular in biological and chemical diagnostics, chip-based systems, known as microarrays, are increasingly relied upon in particular in the detection of pathogens, such as, for example, viruses. Chip-based investigation methods allow simultaneous detection of a multiplicity of chemical or biological reactions in the smallest space. In this manner, on the one hand the requirement for cost-intensive laboratory equipment can be saved, and on the other hand a multiplicity of measurements can be carried out at the same time, so that even the results of complex investigations as a rule are available promptly.
Such chips which are suitable for chemical and biological investigation methods, in particular medical investigation methods, conventionally have a plurality of measurement regions, each of which is functionalized with different chemical or biological molecules so that different chemical reactions can take place and be detected in the individual measurement regions.
A chip having a multiplicity of measurement regions is known, for example, from U.S. Patent Application Publication 2009/0131278 A1. This is a silicon-based chip on the surface of which a multiplicity of electrode pairs is arranged by metallising and structuring. In each case, one electrode pair is present in a measurement region and the measurement regions are arranged in a chequered manner in one plane and form an array.
The electrode pairs each comprise microstructured finger electrodes intermeshed with one another, so that the electrodes in each case have adjacent interfaces over wide regions. The individual measurement regions are separated from one another by mechanical barriers in the form of small walls between the individual measurement regions, so that the measurement regions are present in the form of small compartments or cavities.
The individual measurement regions in such chips or microarrays are conventionally functionalized individually, in the context of chemical or biological diagnostics usually with biologically active substances. The biologically active substances can be, for example, antibodies which react chemically with specific antigens.
The chemical reaction can change the electrochemical relationships in the particular measurement region, so that the chemical reaction is detectable electrically by the electrode arrangement.
However, it is also possible that the molecules applied to the chip surface—capture molecules—react with specific target molecules, in particular bind these chemically, and the compound formed in this way is subsequently reacted chemically, this subsequent chemical reaction being detectable with the electrode arrangement.
The functionalizing is conventionally carried out by what is known as a spotting process, in which each measurement region is treated with a different solution or dispersion of a functionalizing reagent. Water is conventionally used as the solvent or dispersing agent for the functionalizing reagent, so that the measurement regions as a rule are formed hydrophilically, but at least are not hydrophobic.
The functionalizing reagent is immobilized on the chip surface by chemical reactions with the chip surface in the measurement regions or with the electrodes.
In order to obtain a result which is as unambiguous and reproducible as possible by using the chip, it is essential that the various liquids used during the spotting process are not mixed with one another, but each remain by themselves in the envisaged measurement region.
The mechanical barriers provided between the individual measurement regions in the above-mentioned U.S. Patent Application Publication 2009/0131278 A1 usually cannot prevent liquid from passing over from one measurement region into another, since due to the small extent of the individual measurement regions, which is in the micrometer range, and the relatively small width of the barrier between the individual measurement regions in combination with the high surface tension of the water, the liquids mix with one another.
In order to counteract this disadvantage, International Patent Application Publication WO 2014/191114 A2 proposes providing the regions between the individual measurement regions with a hydrophobic coating, in particular with a monolayer based on a perfluoroalkylsilane. Improved results in the spotting process can in fact be achieved in this manner, although the formation of a hydrophobic monolayer on the chip leads to problems in the actual measurement, especially if the chip has to be wetted over a large area with several reagents in succession for the detection. In particular, those reactions in which a substance, for example an antigen, is bound to the substrate, for example an antibody, bonded to the chip surface and the compound formed must then be reacted again for the detection are difficult to carry out with the system described. Individual separate vessels on the chip surface would be advantageous here, also because of the larger sample volume.
U.S. Pat. No. 6,779,637 B2 describes a system for producing hydrophilic measurement regions and hydrophobic intermediate regions, wherein in this case also the regions between the individual measurement regions are said to be raised as little as possible.
Highly hydrophilic measurement regions moreover have the disadvantage that, in particular with an otherwise hydrophobic chip surface, the spotting process can indeed be carried out very precisely and cleanly, but complete removal of individual reagent or sample solutions if the chip surface is wetted several times is possible only with difficulty.
Measurement methods are often sought in which a chip can be treated with several reagent or sample solutions, for example with different solutions of several amplification products for detection of a virus, wherein the specific antibodies present in different measurement regions each bind to particular antigens. Between each wetting of the chip with a solution containing amplification products the chip is cleaned such that all residues of the previous amplification product solution are removed from the chip surface. After the chip surface or the measurement regions have been wetted with the amplification product solutions, a developer solution which reacts with the particular compounds or conjugates in the measurement regions is then applied to the chip surface. This reaction can be detected by the electrodes. It is advantageous here for the chip and in particular the measurement regions to be covered such that the individual measurement regions form closed vessels. A particularly high reproducibility of the results is ensured in this way.
A chip would thus be desirable for which the fluid properties are developed such that on the one hand clean spotting of the individual measurement regions is possible, and on the other hand the chip can be wetted several times in sequence with different reagents, wherein the reagents in each case can be removed again without residue before wetting is carried out with a further reagent. Such properties depend not only on the material of the chip or of the measurement regions and of the mechanical barriers or coatings defining the measurement regions, but rather also on the concrete dimensions of the chip and of the measurement regions, in particular the depth and the diameter of the measurement regions and the spacing of the individual measurement regions.
The prior art thus lacks a concrete configuration of the measurement regions in combination with suitable materials in order to ensure not only defect-free spotting of the individual measurement regions but rather also an improved and reproducible measurement on the chip.